Multiphoton Imaging Core

Advanced imaging and physiology resources for common use

NINDS Institutional Center Core Grant to Support Neuroscience Research: Multiphoton Imaging (MPI) Core

OUR MISSION

The goal of the Multiphoton Imaging (MPI) Core in the Department of Neuroscience at Johns Hopkins is to provide instrumentation for analyzing protein localization, protein dynamics, and protein-protein interactions with high resolution. This facility also allows users to perform time-lapse imaging of multiple fluorophores in living cells and tissues, and to combine high-resolution imaging of fluorescently tagged proteins or ion indicator dyes with electrophysiological monitoring of electrical activity.

The Multiphoton Imaging Core promotes interactions among a diverse group of neuroscientists at Johns Hopkins University. Such interactions have facilitated the investigation of key issues in basic and clinical neuroscience, as illustrated in our List of Publications of work supported by the MPI Core.

FINANCIAL SUPPORT AND MEMBERSHIP

The MPI Core is supported primarily by a P30 Center grant from the NINDS. The PI of this P30 Center is Alex Kolodkin (kolodkin@jhmi.edu), the Director of the MPI Core is Dwight Bergles (dbergles@jhmi.edu), and the Manager of the Core is Michele Pucak (mpucak1@jhmi.edu). Activities in the MPI Core are supported by Terry Shelly, Senior Design Technician at JHU, who fabricates custom instrumentation for Core use. The MPI Core primarily supports the research activities of thirteen JHMI faculty, members of the Neuroscience and Neurology Departments, who constitute the Center Primary Investigators of the P30 grant. However, instrumentation within the MPI Core is also available to other NINDS funded investigators and other investigators at JHU who are involved in neuroscience-related research.

Applications for use of Core facilities are carefully evaluated by Core personnel and directors. Services are allocated based on instrument availability and the guidelines that govern this NINDS Core funding mechanism. Members of Center Primary Investigator laboratories are given priority access to Core instrumentation during particularly busy times. To ensure that Center Investigators' access to equipment is not unduly limited, we are unable to grant membership to laboratories whose research falls outside NINDS research goals. However, we are always happy to provide help with experimental design, approaches for analysis, and in some occasions, access to Core instrumentation for the purpose of performing pilot studies.

Because NINDS requires that P30 Centers participate in cost recovery, an annual MPI Core membership fee is assessed to all users, whether or not they are Center Primary Investigators, the first time they use the MPI Core Facility. This annual fee is based on PI seniority: $2000 for Full Professors, $1250 for Associate Professors, and $750 for Assistant Professors. Fees are used to offset the costs of service contracts and to expand Core capabilities. The annual fee provides users with access to all equipment located in the MPI Core (microscopes and image processing workstations). There is no additional hourly fee assessed to users, and members are not limited with respect to the number of hours/year the laboratory may use the MPI Core. At the end of a laboratory's year of membership, the PI is contacted to determine whether they wish to renew for an additional year.

Please direct any questions regarding access to the MPI Core to the MPI Core manager, Michele Pucak, mpucak1@jhmi.edu.

LOCATION

The Multiphoton Imaging Core is located in Room 1008A, Preclinical Teaching Building, 725 North Wolfe Street, on the Johns Hopkins University East Baltimore campus.  The phone number is 410-502-7858.

RESEARCH SUPPORT

A great deal of expertise is available to users of the MPI Core. Michele Pucak, PhD (MPI Core Manager) has experience in single and multiphoton imaging, immunohistochemistry, maintenance of animals for in vivo imaging, and electrophysiological techniques. The MPI Core Director, Dr. Dwight Bergles, has extensive expertise in imaging and electrophysiological techniques. In addition, the many users of the facility each bring unique knowledge and experience that can be leveraged for the particular experimental needs of investigators.

The first step for all faculty, postdocs, and graduate students who use the MPI Core is a one-on-one orientation on the relevant piece of equipment with the Core Manager. The individual nature of these sessions allows the Manager to provide training appropriate to the background, expertise, and research question of each individual. Depending on these factors, the orientation may simply cover proper use of the equipment; however, it may also include information on choice of fluorophore, immunohistochemical techniques, and proper imaging parameters, as well as the differences between epifluorescence, confocal, and two-photon microscopy. In addition, because the Core offers a variety of options for image analysis (Imaris and Neurolucida), the Core Manager often works with individuals to help them determine what factors to consider when designing the workflow for quantification of their data.

The MPI Core further contributes to educating our community of neuroscientists about imaging and image analysis by organizing demonstrations of equipment, facilitating and publicizing seminars of interest to the imaging field, and hosting meetings to promote sharing of information on such topics as in vivo imaging, tissue clearing, and image processing.

CORE SERVICE

Confocal Microscopy

The facility contains four confocal microscope systems that allow high-resolution imaging of labeled cell components in three-dimensional space. 

Zeiss LSM 800

Our Zeiss LSM 800 is typically used for imaging fixed slides, especially when tiling is required, or for the imaging of live tissue.  It consists of an inverted AxioObserver microscope equipped with three GaAsP detectors. These provide enhanced sensitivity compared to traditional PMT detectors, resulting in decreased bleaching and phototoxicity. The 800 uses filter-free variable spectral dichroics, allowing the user to define specific emission collection parameters rather than being limited to a particular set of installed emission filters.  Because this system is equipped with 405, 488, 561, and 640 nm lasers, users can collect images from tissue stained with a wide variety of fluorophores ranging from DAPI to far-red.  Wide-field DIC images can also be collected using the ECID detector.  The motorized stage, in combination with software features, allows users to perform time-lapse imaging with flexible control of experiment design. The high reproducibility of this stage, combined with software control of acquisition and with post-acquisition processing, facilitates the collection of seamless tiled images.  The 800 also includes an AiryScan detector, which uses photon reassignment to provide enhanced sensitivity and spatial resolution (~ 140 nm laterally and 400 nm axially).  This capability is particularly useful for applications that require the resolution of small objects.  Available objectives are 2.5x air, 10x air, 20x air, 40x oil, and 63x oil.  A second 20x air objective is a long working-distance lens which can be used with a wider array of specimen types.  A separate server is available for off-line processing of acquired images.   The full complement of available Zen software from Zeiss is present, allowing users to use this platform to perform a wide array of experiments, including FRET and FRAP.  

Zeiss LM 880

The Zeiss LSM 880 can be used to image fixed slides, live tissue, or intact mice.  This confocal consists of an upright AxioExaminer.Z1 microscope equipped with three internal detectors - two traditional PMTs and a very sensitive GaAsP detector which reduces the risk of bleaching or phototoxicity. The filter-free spectral detection allows the user to define emission collection parameters, rather than being limited to a particular set of installed emission filters.  Because the 880 is equipped with 405, 458, 488, 514, 561, and 633 nm lasers, users can collect images from tissue stained with a wide variety of fluorophores ranging from DAPI to far-red.  Wide-field DIC images can also be collected using the transmitted light detector.  The motorized stage, in combination with software features, allows users to perform time-lapse imaging with flexible control of experiment design. The height of this motorized stage can be changed to accommodate a variety of sample types, from fixed slides to live mice. The 880 also includes an AiryScan detector, which uses photon reassignment to provide enhanced sensitivity and spatial resolution (~ 140 nm laterally and 400 nm axially).  This capability is particularly useful for applications that require the resolution of small objects.  In addition, the AiryScan detector can be used in a Fast mode to enable rapid collection during time-lapse imaging.  Available objectives are 5x air, 10x air, 25x oil, 40x oil, 63x oil, and 100x oil.  A separate server is available for off-line processing of acquired images.   The full complement of available Zen software from Zeiss is present, allowing users to use this platform to perform a wide array of experiments, including FRET and FRAP.  

In addition to the visible lasers listed above, the 880 confocal is coupled to a near-infrared (IR) tunable pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II), which is positioned on the same table as the 710 (see below), allowing the two systems to share the IR beam from this laser. The IR laser of the two-photon system facilitates imaging of thick specimens, and is capable of imaging fluorescent dyes or proteins in vivo. For two-photon applications on the 880, an additional Zeiss binary GaAsP (BIG) detector with band-pass emission filters permits sensitive detection of red or green fluorophores from deep within tissue. This detector is particularly critical for in vivo applications, and has allowed users to monitor cell motility in intact preparations. Because the BIG detector is extremely sensitive to ambient light, we have constructed a light-tight enclosure around this system, so that both microscopes in this room can be used simultaneously. For in vivo imaging, we have three long working distance, high numerical aperture dipping objectives (10x and 20x lenses which are not coverslip-corrected as well as a 20x coverslip collected lens). 40x and 63x dipping lenses are also available.

Zeiss LSM 710

The Zeiss 710 confocal is most frequently used for imaging live mice or for performing slice imaging, including calcium imaging. It consists of an upright AxioExaminer.Z1 with internal PMTs which have good detection capabilities due to refinements of the light path within the scan head. The filter-free spectral detection allows the user to define emission collection parameters, rather than being limited to a particular set of installed emission filters. Because the 710 is equipped with 458, 488, 514, 543, and 633 nm lasers, users can collect images from tissue stained with a wide variety of fluorophores ranging from GFP to far-red. Wide-field DIC images can also be collected using the transmitted light detector. A variety of custom-made stages are available, permitting the use of a wide range of specimen types. The full complement of available Zen software from Zeiss is present, allowing users to use this platform to perform a wide array of experiments, including FRET and FRAP.

In addition to the visible lasers listed above, the 710 confocal is coupled to a near-infrared (IR) tunable pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II), which is positioned on the same table as the 880 (see above), allowing the two systems to share the IR beam from this laser. The IR laser of the two-photon system facilitates imaging of thick specimens, and is capable of imaging fluorescent dyes or proteins in vivo. For two-photon applications on the 710, an additional Zeiss binary GaAsP (BIG) detector with band-pass emission filters permits sensitive detection of red or green fluorophores from deep within tissue. This detector is particularly critical for in vivo applications, and has allowed users to monitor cell motility in intact preparations. Because the BIG detector is extremely sensitive to ambient light, we have constructed a light-tight enclosure around this system, so that both microscopes in this room can be used simultaneously. For in vivo imaging, we have three long working distance, high-NA dipping objectives (10x and 20x not coverslip-corrected, and 20x coverslip collected). 40x and 63x dipping lenses are also available.

Thorlabs Bergamo confocal

Many investigators want to resolve neural activity using genetically encoded calcium and voltage sensors, which requires rapid image acquisition. Resonant scanning galvanometers provide a substantial improvement in full frame image acquisition, allowing video rate capture (30/s) at a resolution of 512 x 512 pixels. It has been reported that resonant scanning also reduces the impact of brain motion and provides more efficient excitation of fluorophores, due to the reduced dwell time of the laser during scanning. To implement this technology, we have established a strategic partnership with Thorlabs. They have provided the MPI Core with a Bergamo II Scope equipped with both galvo/galvo and resonant/galvo scanners. This system has several other capabilities that are not currently available in the core, including a 473 CW blue laser for focal uncaging. The entire scan head is mounted on a rotatable platform, enabling lateral imaging without rotating the head of the animal.  

Additional Equipment for Confocal Imaging

Custom, interchangeable temperature-controlled stages are available for use in live cell/tissue imaging experiments. Tissue superfusion is achieved using a gravity-fed system that passes through computer-controlled solenoid valves. Solutions can be oxygenated and, if necessary, heated using an in-line heater or a peltier chamber heater, depending on the application.

Support for in vivo applications is also available, including isoflurane vaporizers, anesthesia induction chambers, and temperature-controlled warming pads.

A piezoelectric focus drive is available when rapid z-focus control is required. Piezoelectric positioning drives provide higher focusing speed (~10 vs 100 msec) and better resolution (~1 vs. 100 nm) than stepper motor-drives. This is especially advantageous for applications such as tracking mitochondrial movement and assessing process motility of labeled cells in vivo, where the ability to assess temporal changes is limited by the time required to construct high-quality z-stack images.

Electrophysiological Components

Whole cell current and voltage clamp recordings can be performed using an Axon Instruments Multiclamp 700B amplifier. Acquisition is controlled by a Digidata 1322A digital-to-analog converter, and a Pentium 4 PC computer running pClamp10 and Origin analysis software. Additional equipment such as a secondary amplifier (Brownlee), a timer/stimulator (A.M.P.I), black-and-white monitor, camera controller, and halogen power supply are positioned in a moveable rack with castors, allowing this system to be moved into position near whichever microscope best suits an investigator's needs.

Epifluorescence Imaging

Zeiss Cell Observer

The Zeiss Cell Observer system consists of an AxioObserver inverted microscope equipped with fluorescent and transmitted light, an Axiocam MRm, and filter sets compatible with DAPI, GFP, Cy3, and Cy5. Available objectives are 5x, 10x, 20x, 40x, and 63x. This microscope is often used for long-term imaging of cells because it is equipped with an environmental chamber to permit control of temperature, humidity, and CO2. A motorized stage facilitates the identification and imaging of multiple regions of interest, and the Experiment Designer software module permits flexible design of experiments and provides various post-processing capabilities.

The system can also be used to image fixed slides. One feature that is useful to many labs is the motorized stage, which supports acquisition of tiled images and allows users to capture large regions of interest at high magnification. Post-processing features are available to remove line artifacts that are often apparent in such tiled images.

Keyence Microscope

The Keyence is a benchtop widefield inverted microscope that is used to image fixed slides.  It can also be used with an insert within which temperature, humidity, and CO2 can be controlled, providing the option of performing long-term imaging of live cells. Both fluorescent and transmitted light are available, and filter sets are compatible with DAPI, GFP, CY3 and Cy5.  In addition to the very sensitive monochrome camera, the Keyence contains a color camera that is used for imaging specimens containing cresyl violet or similar dyes. A motorized stage facilitates the identification and imaging of multiple regions of interest and also allows the collection of tiled images. This microscope also has the ability to use structured illumination (SIM) to improve spatial resolution.

Stereo Zoom Microscope

Our Zeiss AxioZoom microscope provides the advantages of a stereomicroscope - zoom optics and long working distance - with the high resolution of a light microscope. The microscope has two high numerical aperture zooms lenses which, combined with the motorized zoom, allow quick and easy switching through the entire zoom range of 7x-260x.

The microscope is equipped with both fluorescent and transmitted light. Investigators can view and image large samples with excellent brightness and resolution without the need for tiling and stitching. In addition, the long working distance means that the microscope can aid in the dissection of small tissue samples. Finally, this microscope is also use to perform in vivo imaging of transgenic mice.

Image Analysis

Imaris

The Core has two dedicated image analysis workstations each running Imaris (Bitplane) software, which is designed to provide automated quantification of fluorescent three-dimensional images.  Imaris can perform isosurface rendering, object detection/counting, filament tracing/particle tracking, and quantitative co-localization; these operations can be used to extract a wide array of quantitative information from images.  Additionally, by interfacing with Matlab, the ImarisXT module allows users to develop their own task-oriented algorithms, or to use analysis modules developed by others. The Imaris suite of programs is compatible with most standard formats, include Tiff series, BMP series, and images collected with Zeiss, Leica, and Olympus acquisition software. Our service contract with Bitplance ensures that we always have access to the most recent updates and improvements to the program; furthermore, this contract provides access to a knowledgeable representative who can help with determining the most efficient workflow for image quantification on an individualized basis.  

Neurolucida

Additional image analysis capabilities are provided by our Neurolucida Neuron Tracing system (Microbrightfield). This system offers a variety of drawing/analysis options, including cell tracing/morphology quantification, 3D brain mapping, and serial section reconstruction. The software is paired with a Zeiss AxioImager microscope equipped with transmitted light and a motorized stage, to allow tracing directly from slides. Alternatively, image files collected on another system (including confocals) can be imported into the Neurolucida software. The analysis capabilities of Neurolucida complement those which can be achieved using the Imaris suite of programs, because the latter is geared almost entirely toward analysis of fluorescent images, whereas Neurolucida excels at reconstruction and analysis of brightfield images (e.g., DAB labeled cells). Because this system includes a color camera, it also provides a route for obtaining color images of brightfield samples, such as LacZ.

Autoquant

Autoquant is a deconvolution software package that produces extremely high-quality results for both widefield and confocal images.

Zen

Both the 800 and the 880 confocal computers are directly connected to servers which provide the entire complement of post-acquisition processing available in the Zen software from Zeiss. This enables processing to be done off-line, freeing time on the confocals for image acquisition.

Stereotaxic Surgery Station

Both the 800 and the 880 confocal computers are directly connected to servers which provide the entire complement of post-acquisition processing available in the Zen software from Zeiss. This enables processing to be done off-line, freeing time on the confocals for image acquisition.

The Angle2 stereotaxic system (Leica) consists of a software-assisted stereotaxic instrument that facilitates the accurate targeting of brain regions by using software combined with transponders that detect the position of the pipette carrier. Advantages include an on-screen atlas to facilitate identification of targets, the ability to use an angled approach without the need for path-of-approach calculation, and the ability to automatically correct for head tilt.

3D Printer

A MakerBot Replicator 2x allows individuals to use software to generate a 3D model of their desired product; this is then converted into file formats the MakerWare software can output to the 3D printer. This equipment permits inexpensive and efficient production of a wide variety of research components.

Application for Service

Researchers wishing to use the Multiphoton Core must first complete the Application for Service and forward to the Core Manager, Michele Pucak (mpucak1@jhmi.edu). Once approved, arrangements for an orientation session will be made. Access to the Core will not be granted without an approved application and all users must complete an orientation session.

Equipment Signup

Procedures for reserving equipment will be reviewed in an orientation session required prior to equipment access. Upon completion of the orientation session and a preliminary training period, equipment is also available on a walk-in basis. Links to reservations calendars are below.

Contact Information

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